A key challenge of current aging research is to identify strategies for healthy aging in which mid-life vigor is prolonged. We use the powerful genetic model system C. elegans to decipher how genes influence healthspan and to identify molecular ways to combat the general functional decline that accompanies aging. microRNAs (miRNAs) are small molecules that target partially homologous mRNA transcripts to down-regulate their expression via a prevalent and conserved gene regulatory mechanism. miRNA roles in aging are just beginning to be addressed, but initial work indicates that expression of many C. elegans miRNAs changes during adult life (in fact there is a major mid-life decline crisis in miRNA levels in this animal) and that many miRNAs may impact the quality of aging, or healthspan. Characterizing this major biological component of middle-age decline will be critical for the overall effort of understanding how gene networks interact to influence aging quality. We therefore propose to test how extensively the conserved microRNAs of this one animal influence healthspan at the tissue and organism levels. We will also study in detail the mechanisms by which two miRNAs with mammalian homologs involved in processes relevant to problems in human aging modulate nematode healthspan. A goal is to document some of the first examples in which manipulation of miRNA expression in vivo might be exploited to extend healthspan. We propose two aims: Aim1: To establish a genomic overview of how the conserved miRNAs of C. elegans impact the biology of aging. We will characterize aging phenotypes of C. elegans mutants of mir genes that are conserved between nematodes and humans, studying locomotory decline, rate of lipofuscin accumulation, pharyngeal muscle pumping rate, and thermotolerance. Data will provide an overview of how conserved miRNAs impact healthspan and may identify key node miRNA modulators of the process. Aim 2: To probe mechanisms by which two miRNAs that are differentially regulated in middle-age alter healthspan, and to model miRNA interventions that increase the quality of aging. We will focus on mechanistic dissection of healthspan-promoting miRNA mir-34, which increases in concentration in middle age, and healthspan-promoting mir-45, which decreases in middle age. The mammalian counterpart of mir-34 is documented to have tumor suppressor activity; the mammalian counterpart of mir-45 influences formation of structures implicated in brain synaptic plasticity. Our studies will include detailed phenotypic analyses of healthspan phenotypes, modulation of expression of specific miRNAs in whole-animal context to alter healthspan outcomes, and specific tests of working models for bioactivity in aging. Overall, the studies we propose address a major gap in our understanding of the genes that impact aging. Our findings might be extrapolated to influence experiments in higher organisms that may ultimately inspire design of healthspan-extending interventions. PUBLIC HEALTH RELEVANCE: Our interest is in genes that promote healthy and robust aging, a topic central to the mission of the National Institute on Aging. Our work, and that of others, has implicated microRNAs--small non-coding RNAs that down-regulate expression of target mRNA transcripts--in the aging process. We have found that ~ half of C. elegans miRNAs change in abundance over adult life, with most lowering in concentration in middle age. Because miRNAs are proposed to play an important role in biological robustness (which diminishes with age), we speculate that miRNAs as a general class will have a large impact on healthy aging. As we screen miRNA mutants for their influence on aging biology and characterize specific mechanisms of miRNA action, we will show how these small molecule regulators might be used to extend the period of healthy aging in a physiological setting. Indeed, at least one miRNA on which we will focus mechanistic studies appears to regulate dietary restriction metabolism-a conserved healthy state characterized by increased vigor, decreased incidence of age-associated disease, and extended lifespan. Our findings may ultimately inspire design of healthspan-extending interventions in humans.